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Mutant Glucose Dehydrogenase

Inactive Publication Date: 2012-05-03
ARKRAY INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]The present inventors intensively studied for attaining the above-described object to discover that, as compared to the CyGDH (QY), the substrate specificities are further improved by replacing the sites corresponding to positions 326 and 365 of amino acid residues of its α-subunit constituting the CyGDH with glutamine and tyrosine, respectively, and further replacing the site corresponding to position 472 with tyrosine, and that, also in GDH homologues, similar effect can be obtained by replacing the sites corresponding to positions 326, 365 and 472 with glutamine, tyrosine and tyrosine, respectively, thereby completing the present invention.

Problems solved by technology

The wild-type CyGDH and PQQGDH have a drawback in that they are incapable of accurately measuring the blood sugar level in the case where the blood maltose level of patients is high, because they react not only with glucose but also with maltose.
Especially in Japan and the United Kingdom, maltose is used as an energy material in infusion solutions, and, in fact, there have been some accidents caused by the wrong measurement results wherein patients who received administration of such infusion solution by peritoneal dialysis or the like and whose blood sugar level was low were mistaken for high blood sugar level due to a sensor that uses a PQQGDH for measuring the blood sugar level.
However, the effect to avoid the influence of maltose was not perfect.

Method used

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  • Mutant Glucose Dehydrogenase
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  • Mutant Glucose Dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

example 1

Plasmids Expressing GDH or CyGDH of Burkholderia cepacia

[0057]As a plasmid expressing a GDH of Burkholderia cepacia, a plasmid expressing an α-subunit and a γ-subunit of the GDH was prepared; and, as a plasmid expressing a CyGDH, a plasmid expressing an α-subunit, a β-subunit and a γ-subunit was prepared.

Plasmid Expressing α-Subunit and β-Subunit of GDH

[0058]As a plasmid expressing an α-subunit and a γ-subunit, a plasmid pTrc99A / γ+α which is described in WO02 / 036779 (corresponding to EP1331272A1, US2004023330A1 and CN1484703A) was used. This plasmid is a plasmid wherein a DNA fragment contiguously containing a GDH γ-subunit structural gene and a GDH α-subunit structural gene, which was isolated from chromosomal DNA of Burkholderia cepacia KS1 strain (FERM BP-7306), is inserted into the NcoI / HindIII site which is a cloning site of the vector pTrc99A. The GDH γα gene in this plasmid is regulated by the trc promoter. The pTrc99A / γ+α has an ampicillin resistance gene.

[0059]The whole p...

example 2

Search of Substrate Interaction Site by Introducing Mutations into CyGDH α-Subunit Gene

[0065](1) Introducing Mutations into Positions 326, 365 and 472

[0066]Mutations were introduced into the GDH α-subunit gene contained in the pTrc99Aγαβ obtained in the Example 1 so that serine residue at position 326, serine residue at position 365 and alanine residue at position 472 of the α-subunit that was coded for by that gene were replaced with other amino acid residues.

[0067]Specifically, the codon corresponding to the serine at position 326 (TCG), the codon corresponding to the serine at position 365 (TCG) and the codon corresponding to the alanine at position 472 (GCG) of the GDH α-subunit genes contained in the plasmids pTrc99A / γ+α and pTrc99Aγαβ as described in Example 1 were replaced with codons corresponding to other amino acids using a commercially available site-specific mutagenesis kit (Stratagene, QuikChangeII Site-Directed Mutagenesis Kit).

[0068]The sequences of forward primers an...

example 3

Analysis of Substrate Specificities of Mutant GDHs

[0074]Mutant GDHs were produced by using the plasmids expressing mutant GDHs which were obtained in the Example 2, and their substrate specificities were studied.

(1) Culture

[0075]The cells of Escherichia coli DH5α strain wherein mutations were introduced in various patterns were each cultured under shaking at 37° C. overnight in 2 ml of LB medium (containing 50 μg / ml of ampicillin and 30 μg / ml of kanamycin) by using a L-shaped tube. These cultures were inoculated into a 500 ml Sakaguchi flask that contained 150 ml of LB medium (containing 50 μg / ml of ampicillin and 30 μg / ml of kanamycin), and the resultants were cultured under shaking at 37° C. Three hours after the beginning of the culturing, IPTG (isopropyl-β-D-thiogalactopyranoside) was added so as to attain a final concentration of 0.1 mM, and the resultants were cultured for another 2 hours.

(2) Preparing Crude Enzyme Samples

[0076]The bacterial cells were collected from the cultu...

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Abstract

A mutant glucose dehydrogenase having an amino acid sequence at least 80% identical to SEQ ID NO:3 and having glucose dehydrogenase activity, wherein amino acid residues corresponding to positions 326, 365 and 472 of said amino acid sequence are replaced with glutamine, tyrosine and tyrosine, respectively, and wherein said mutant glucose dehydrogenase shows an improved substrate specificity to glucose and a reduced reactivity to disaccharides.

Description

TECHNICAL FIELD[0001]The present invention relates to a mutant glucose dehydrogenase showing an improved substrate specificity. Specifically, the present invention relates to a glucose dehydrogenase comprising a mutant-type α-subunit which is produced by introducing mutations into amino acid residues of its α-subunit that constitutes a cytochrome C-containing glucose dehydrogenase (hereinafter also referred to as CyGDH), and relates to a gene thereof. The glucose dehydrogenases of the present invention can be suitably used for glucose sensors, glucose assay kits and so forth, and are useful in the fields of biochemistry, clinical medicine and so forth.BACKGROUND ART[0002]At present, a wild-type CyGDH, a PQQGDH using pyrroloquinoline quinone as a coenzyme, or the like is used for self-monitoring blood glucose sensors. The wild-type CyGDH and PQQGDH have a drawback in that they are incapable of accurately measuring the blood sugar level in the case where the blood maltose level of pat...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N1/15C12N1/21C12N1/19C12N9/96C12N15/53
CPCC12N9/0006C12Q1/006
Inventor SODE, KOJIKOJIMA, KATSUHIRO
Owner ARKRAY INC
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